One gene, two phenotypes – understanding the pathomechanics and leukemia development in congenital neutropenia and cyclic neutropenia

一种基因，两种表型——了解先天性中性粒细胞减少症和周期性中性粒细胞减少症的病理机制和白血病发展

• 批准号: 455056283
• 负责人: Professorin Dr. Julia Skokowa, Ph.D.
• 金额: --
• 依托单位: Innere Medizin II: Onkologie, Hämatologie, Klinische Immunologie, Rheumatologie und Pulmologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/455056283?language=en
• 关键词: gene; two; phenotypes; understanding; pathomechanics

Severe congenital neutropenia (CN) is a mono-lineage pre-leukemia bone marrow failure syndrome, characterized by early onset of neutropenia accompanied by severe infections. CN is a heterogeneous disease, caused by mutations in different genes, but most commonly in ELANE, encoding the neutrophil elastase (NE) protein, which is inherited as an autosomal dominant disease. A second form of hereditary neutropenia is cyclic neutropenia (CyN), which is characterized by oscillations of the neutrophil counts with a nadir occurring every 21 days. During the periods of low neutrophil counts, patients may suffer from infections, such as aphthous stomatitis, periodontitis and typhlitis. Almost all cases of CyN result from mutations in ELANE and, remarkably, the mutations causing CyN invariably overlap with those described in CN. It is unclear how the same mutation can cause CN and CyN. One severe complication of CN, and to a much lesser extent of CyN, is the development of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Granulocyte-colony stimulating factor (G-CSF) is a life-saving drug for CN and CyN patients, improving the granulopoiesis and preventing severe infections. However, the risk of leukemia development in CN directly correlates with the G-CSF dose required. The cumulative incidence of MDS or AML in CN is around 20% after 10 years of G-CSF treatment. Patients with CyN have a much lower but existent susceptibility to develop MDS and AML. It is unclear why leukemia progression is much lower in CyN patients. No mouse model exists to study CN and CyN caused by ELANE mutations, as targeting the mouse ortholog of ELANE in mice does not cause neutropenia. In addition, as CN and CyN are often caused by identical mutations in ELANE, it is not possible to generate accurate models that will differentiate between the two disorders by simple gene editing methods. Therefore, we aim to generate a model for CN and CyN caused by ELANE mutations and for the malignant transformation in CN, using patients-derived induced pluripotent stem cells (iPSCs). Importantly, iPSCs carry the full genetic background of the patients, and will therefore include any possible genetic modifiers that have not been identified so far. We recently published the generation and granulocytic differentiation of patients-derived iPSCs, including for CN caused by mutations in ELANE. iPSCs recapitulated the defective granulopoiesis observed clinically. No published study utilized an iPSCs model of CyN. We plan to compare pathomechanics of CN and CyN using patients-derived iPSCs. In addition, as CN patients carry a high risk for leukemic transformation, related to acquired G-CSFR mutations, we aim to generate a model for pre-leukemia by introducing these mutations using CRISPR/Cas9. We believe that this model will help elucidate the pathways involved in early malignant transformation processes in CN and may reveal more general pathways contributing to malignant transformation.

严重先天性中性粒细胞减少症（CN）是一种单系白血病前期骨髓衰竭综合征，其特征是早期出现中性粒细胞减少症并伴有严重感染。 CN 是一种异质性疾病，由不同基因突变引起，但最常见于编码中性粒细胞弹性蛋白酶 (NE) 蛋白的 ELANE，该蛋白作为常染色体显性遗传病遗传。遗传性中性粒细胞减少症的第二种形式是周期性中性粒细胞减少症 (CyN)，其特征是中性粒细胞计数波动，每 21 天出现一次最低点。在中性粒细胞计数较低期间，患者可能会遭受感染，例如口疮性口炎、牙周炎和盲肠炎。几乎所有 CyN 病例都是由 ELANE 突变引起的，值得注意的是，引起 CyN 的突变总是与 CN 中描述的突变重叠。目前尚不清楚相同的突变如何导致 CN 和 CyN。 CN 的一种严重并发症（CyN 的严重程度要小得多）是发生骨髓增生异常综合征 (MDS) 和急性髓系白血病 (AML)。粒细胞集落刺激因子（G-CSF）是 CN 和 CyN 患者的救命药物，可改善粒细胞生成并预防严重感染。然而，CN 发生白血病的风险与所需的 G-CSF 剂量直接相关。 G-CSF治疗10年后，CN中MDS或AML的累积发生率约为20%。 CyN 患者发生 MDS 和 AML 的可能性要低得多，但仍然存在。目前还不清楚为什么 CyN 患者的白血病进展速度要慢得多。目前尚无小鼠模型可用于研究 ELANE 突变引起的 CN 和 CyN，因为针对小鼠体内 ELANE 的直系同源物不会引起中性粒细胞减少症。此外，由于 CN 和 CyN 通常是由 ELANE 中的相同突变引起的，因此不可能通过简单的基因编辑方法生成区分这两种疾病的准确模型。因此，我们的目标是使用患者来源的诱导多能干细胞（iPSC）建立由 ELANE 突变引起的 CN 和 CyN 以及 CN 恶性转化的模型。重要的是，iPSC 携带患者的完整遗传背景，因此将包含迄今为止尚未发现的任何可能的遗传修饰因子。我们最近发表了患者来源的 iPSC 的生成和粒细胞分化，包括 ELANE 突变引起的 CN。 iPSC 再现了临床观察到的粒细胞生成缺陷。尚未发表的研究利用 CyN 的 iPSC 模型。我们计划使用患者来源的 iPSC 来比较 CN 和 CyN 的病理力学。此外，由于 CN 患者具有与获得性 G-CSFR 突变相关的白血病转化的高风险，我们的目标是通过使用 CRISPR/Cas9 引入这些突变来生成白血病前期模型。我们相信该模型将有助于阐明 CN 早期恶性转化过程所涉及的途径，并可能揭示导致恶性转化的更一般途径。